阅读说明：本技术 Gpr115基因在制备抗肺癌药物及其诊断试剂盒中的应用 (Application of GPR115 gene in preparation of anti-lung cancer medicine and diagnostic kit thereof ) 是由 黄剑飞 张筱静 王营静 于 2019-11-05 设计创作，主要内容包括：本发明公开了GPR115基因在制备抗肺癌药物及其诊断试剂盒中的应用,属于癌症精准医疗药物技术领域。本发明通过肺癌临床样本组织芯片,免疫组化证实,GPR115在肺癌中表达增高,且与预后相关；通过体外细胞实验研究siRNA下调GPR115基因表达对肺癌细胞增殖侵袭等生物学行为的影响,发现特异性的siRNA序列可以有效抑制人肺癌细胞株H1650和SPCA中GPR115蛋白的表达,siRNA抑制GPR115表达后,H1650和SPCA细胞的增殖减慢,细胞侵袭能力降低。GPR115作为肺癌基因治疗的靶点,在制备精准医疗的诊断试剂盒和用于治疗高表达GPR115的肺癌中,将具有广泛的应用。(The invention discloses an application of GPR115 gene in preparation of an anti-lung cancer drug and a diagnosis kit thereof, and belongs to the technical field of precise cancer medical drugs. The lung cancer clinical sample tissue chip is used for immunohistochemistry verification, so that the expression of GPR115 in lung cancer is increased and is related to prognosis; the influence of the siRNA for down regulating GPR115 gene expression on biological behaviors such as lung cancer cell proliferation invasion and the like is researched through in vitro cell experiments, and the specific siRNA sequence is found to be capable of effectively inhibiting the GPR115 protein expression in human lung cancer cell strains H1650 and SPCA, after the GPR115 expression is inhibited by the siRNA, the proliferation of H1650 and SPCA cells is slowed down, and the cell invasion capacity is reduced. GPR115 is used as a target point of lung cancer gene therapy, and can be widely applied to the preparation of a diagnostic kit for precise medical treatment and the treatment of lung cancer with high GPR115 expression.)

Application of GPR115 gene in preparation of a kit for lung cancer diagnosis.

Application of GPR115 gene in preparation of a kit for prognosis of lung cancer.

Application of GPR115 gene in preparation of medicaments for treating lung cancer.

4. The use of claim 3, wherein the medicament is designed to target the GPR115 gene.

5. The use of claim 3 or 4, comprising one or more of the following three siRNA sequences:

GPR115-shRNA-1：5′-GAUCCAAGAUUCACCUAAAdTdT-3′；

GPR115-shRNA-2：5′-GGAUUUACAUGUAAUCAAAdTdT-3′；

GPR115-shRNA-3：5′-CAUUGAGAGUGUAGCUCAAdTdT-3′。

6. the use of claim 5, wherein the siRNA sequence is used to inhibit proliferation or invasion of lung cancer cells.

Technical Field

The invention belongs to the technical field of precise cancer medical drugs, and particularly relates to an application of GPR115 gene in preparation of an anti-lung cancer drug and a diagnostic kit thereof.

Background

Lung cancer (Lung cancer) is a highly invasive, rapidly progressing malignant tumor due to uncontrolled growth of Lung tissue cells. According to the latest global cancer statistics, lung cancer remains the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for 80% -85% of the most common. Most patients are often incurable because they are not diagnosed until late, losing therapeutic value or failing to operate. Treatment of lung cancer is closely related to cell type, transmission range and human performance status, and commonly includes: palliative therapy, surgery, chemotherapy, and radiotherapy. The advent of targeted therapy and immunotherapy in recent years has brought good news to the treatment of lung cancer, but the 5-year survival rate of patients is still only 16%. Therefore, the research on the occurrence and development mechanism of lung cancer should be enhanced, and new lung cancer markers are searched to provide new targets and new ideas for the treatment of lung cancer.

The G protein-coupled receptor 115 (GPR 115) is a member of a G protein family (GPCRs), is positioned at 6p12.3 and has a full length of 752aa, and is characterized by containing 7 seven transmembrane domains or topological structures. The activated GPCR acts as a guanine nucleotide exchange factor (GEF) for the alpha subunit of the heterotrimeric G protein, catalyzing the release of GDP and GTP binding to the activated G protein. The activated G protein subunits (α GTP and β γ) can then bind with downstream effectors, thereby modulating cellular physiology and the function of multiple systems, such as the immune system, cardiovascular system, nervous system, reproductive system, and sensory system. Recent data indicate that GPCRs are expressed in cancers of lung, prostate, colon, pancreatic and mesenchymal cells (from the tumor microenvironment) and stimulate cancer cell proliferation, invasion, metastasis, migration, adhesion and angiogenesis. Luo, W, et al, demonstrated that interference with the expression of the G protein-coupled receptor LGR4 inhibited prostate cancer cell migration, invasion and colony formation, and reversed epithelial-to-mesenchymal transition (EMT). GPR115 is one of the members of GPCRs family, and has important significance in tumor and pathology aspects. Wang, j.c. et al found a potential role for GPR115 in the pathogenesis of inflammatory skin diseases and may be related to the therapeutic role of glucocorticoids in these diseases. Interestingly, the study predicted that GPR115 is closely related to methylation and lung cancer treatment via the database. However, this is merely a predictive conclusion of a database and lacks generality and effective experimental demonstration to illustrate. Therefore, the specific expression and mechanism of action of GPR115 in lung cancer remains to be further investigated.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the application of the GPR115 gene in preparing a medicament for treating lung cancer, and meets the requirement of precise medical treatment. Another problem to be solved by the invention is to provide the application of the GPR115 gene in a kit for lung cancer diagnosis or prognosis judgment.

In order to solve the above problems, the present invention adopts the following technical solutions

Use of GPR115 gene (Gene No., Entrez ID: 221393) in the preparation of a diagnostic kit for lung cancer.

Application of GPR115 gene in preparation of a kit for lung cancer prognosis judgment.

Application of GPR115 gene in preparing a medicament for treating lung cancer.

The medicine is designed by taking GPR115 gene as a target point.

The drug comprises one or more of the following three siRNA sequences:

GPR115-shRNA-1：5′-GAUCCAAGAUUCACCUAAAdTdT-3′；

GPR115-shRNA-2：5′-GGAUUUACAUGUAAUCAAAdTdT-3′；

GPR115-shRNA-3：5′-CAUUGAGAGUGUAGCUCAAdTdT-3′。

the siRNA sequence is used for inhibiting the proliferation or invasion of the lung cancer cells.

Has the advantages that: compared with the prior art, the method adopts a tissue chip and an immunohistochemical technology to detect the expression condition of the GPR115 gene in the lung cancer, finds that the expression of the GPR115 protein is obviously increased in the lung cancer tissue, and the prognosis of patients with a GPR115 high expression group is poorer. In addition, through the technologies of Western blot, Transwell chamber and the like, the influence of the downregulation of GPR115 gene expression on biological behaviors such as lung cancer cell proliferation and invasion is researched in vitro tests, and the specific siRNA sequence is found to be capable of effectively inhibiting the expression of GPR115 protein in lung cancer cell strains, so that the lung cancer cell proliferation, migration and invasion capacities are remarkably reduced. GPR115 can be used as a target point of lung cancer cell gene diagnosis and treatment, and can be widely applied to preparation of lung cancer diagnosis kits and therapeutic drugs.

Drawings

FIG. 1 is a graph of the TCGA and Oncoine databases analyzing the expression level of GPR115 mRNA in lung cancer tissues;

FIG. 2 is a graph of IHC detecting GPR115 expression in lung cancer tissue and in paracancerous and normal lung tissue; a1-3 is lung adenocarcinoma, and cancer cell staining is strong positive; b1-3 is lung squamous carcinoma, and the cancer cell staining is strong positive; c1-3 is lung adenosquamous carcinoma, and the staining of lesion cells is strong positive; d1-3 is normal lung tissue, and lung epithelial cells are weakly positive or negative in staining. Wherein A1-D1 are simple immunohistochemical staining pictures, and brown staining cells are GPR115 expression positive cells; A2-D2 typical cancer cell partial magnification picture, in the figure black arrow indicates cancer cell; A3-D3 is an analysis result graph of the multispectral automatic pathology imaging system, brown indicates strong positive (+++), orange color indicates positive (++), yellow indicates weak positive (+), and blue indicates negative (-), all the pictures are taken under a 20X objective lens, and pictures are taken under the same magnification by applying Photoshop;

FIG. 3 is a Kaplan-Meier plotter graph of GPR115 expression, gender, lung cancer differentiation degree and TNM staging on five-year survival rate of lung cancer patients, from left to right, and from top to bottom, respectively, the Kaplan-Meier plotter graph of GPR115 expression, gender, lung cancer differentiation degree and TNM staging on five-year survival rate of lung cancer patients (P < 0.05, the difference has statistical significance);

FIG. 4 is a graph showing the expression of GPR115 in transfected and transfected lung cancer cell lines by Western Blot assay; a is the expression of GPR115 protein in A549, H1650, SPCA and H1975 lung cancer cells; b is the verification of GPR115 interference situation by 3 siRNA fragments, wherein hs-GRP115-siRNA-2 has higher inhibition efficiency (p < 0.05);

FIG. 5 is a graph of proliferation index (. p. < 0.05) for transfected and untransfected lung cancer cells tested at corresponding time points in the CCK-8 assay;

FIG. 6 is a graph of the effect of the Transwell assay on the ability of GPR115 to invade H1650 and SPCA cells (. p < 0.05);

fig. 7 is a graph of the effect of GPR115 on the migratory capacity of H1650 and SPCA cell lines tested by scratch assay (. p < 0.05).

Detailed Description

The present invention will be further described with reference to the following specific examples.

The main reagents used in the following examples are: DAB staining solution kit: new biotechnology of fuzhou mai, china; rabbit anti-human GPR115 monoclonal antibody: Sigma-Aldrich, USA, Cat No. HPA 007158; GAPDH antibody: GOOD HERE, Inc., Hangzhou, China, Cat AB-M-M001; 0.01mol/L citric acid buffer (pH 6.0): china fir bridge, beijing; xylene, neutral gums, and the like are provided by the pathologists; lung cancer cell lines: purchased from shanghai, china CBTCCCAS; PVDP film: millipore corporation, usa, cat # HATF 00010; horse radish peroxidase-labeled goat anti-rabbit/mouse IgG: abcam, Inc. of USA; 5% BSA: shanghai national drug, nantong maije; 1640 medium, fetal bovine serum: thermo scientific, usa; cell cryopreservation solution: suzhou New Saimei Biotechnology, Inc., China; CCK8 kit: dougen Japan, cat # JE 603; luminescent liquid: millipore corporation, USA, Cat WBKLS 0100; matrigel gel: BD incorporated, usa; transwell cell: corning Incorporated, usa; RPMI-1640 complete medium: adding RPMI-1640 at a ratio of 9: 1, mixing with fetal calf serum, and storing at 4 deg.C. 1 × TBST 1L: taking 2.42g of Tris, 8.0g of NaCl and 200.5mL of Tween, mixing and dissolving, using double distilled water to fix the volume to 1L, and storing at normal temperature. 1 × electrophoretic fluid 1L: 3.02g of Tris, 18.8g of glycine and 1g of SDS, adding double distilled water to a constant volume of 1L, mixing uniformly, and storing at normal temperature. 1 × 1L of membrane transfer liquid: 14.4g of glycine and 3.03g of Tris, adding double distilled water, stirring and dissolving, adding 200mL of anhydrous methanol, metering to 1L, and uniformly mixing (preparation in use). 100mL of confining liquid: adding 100mL of 1 XTSST into 5g of skimmed milk powder, and mixing and dissolving (when needed).

The main instruments used in the following examples are as follows: full-automatic pathology imaging system: perkin elmer vectra, usa; a cell culture box: thermo Scientific 8000, usa; electrophoresis apparatus: Bio-Rad corporation, model minirotean 3 cell; electrotransformation appearance: dalian Bianmai Tech Co., Ltd, model PS-9; an enzyme-labeling instrument: thermo corporation, usa, model MK 3; integral type chemiluminescence imager: ChemiScope 5300 Pro; fluorescence inverted phase contrast microscope: olympus, japan; an optical microscope: XDS-1A.